Modified bacteria and toxins, immunizing serums and method of producing them



Patepted Mar. 15,1927.

UNITED STATES PATENT OFFICE.

WINFORID P. LARSON, OF MINNEAPOLIS, MINNESOTA.

MODIFIED BACTERIA AND TOXINS, IMMUNIZING SEBUMS AND METHOD 0] PRO- DUCING THEM.

No Drawing. Application filed November 4, 1985,8er1a1 No. 66,864. Renewed July 80, 1928.

It is the object of my invention to modify bacteria and their products and toxins so that theycan be used as antigens more eflicaciously; and to produce immunizing serums by the use of such modified antigens.

The present application is in part a continuation of my prior application Serial No. 758,955, filed December 30,1924, and of my prior application filed August 27, 1925. 1

If bacteria or bacterial products and toxins,without being modified or detoxified by some method, are used as antigens by being injected into the system of a human being or other animal, they will ordinarily produce either disease ordeath unless intheir available antigenic properties may injected in such small doses that the production of immunity either requires along series of doses or does not occur at all; while if they are treated and modified by methods previously known and practiced to render them non-poisonous or non-diseaseproducing, their power to immunize is either seriously interfered with or destroyed or they produce undesirable sensitization ef fects. This has constituted one of. the principal difficulties in producing immunity against the diseases caused by such bacteria and their products or toxins.

In studying this problem, I have discovered how bacteria, as Well as their poisonous products, usually spoken of as toxins, may be detoxified without materially lessening their antigenic properties and their-power to produce immunity; and, indeed, how

efl'ect be increased. by ma ing' it possible to use safely and eflicaciously larger amount-s of the," antigen. In doing this, I attenuate or detoxify such bacteria or toxins, or both,

by treating them with a soluble salt of one off-the higher fatty acids, which salts are commonly designated as soaps and are all surface-tension depressants, (or capilliary active substances, the two terms being synonomous,) or with a soluble salt of some other organic acid which is surface-tension depressant. The salts I prefer to employ are .derived from fatty acidshaving melting points below 44 C. and are soluble in water at that or a lower, temperature. Of

as a soluble salt of ricinoleic Serial No. 52,963,

these, I prefer the salts derived from unsaturated fatty acids, particularly those having one or more hydroxyl groups,such cially purified castor oil soap. I prefer to work with the castor oil soa solution at a pH of 7.4 to 8.0, within wiich pH range the desired attenuating efi'ect is at a maximum.

Thus, for example, I may attenuate or modify the pathogenicity of such organisms as streptococci, pneumococci or the ultra microscopic bacteria sometimes spoken of as filterable viruses or of such toxins as the diphtheria, tetanus, or scarletfever toxins, by treatingthem in this manner. a

The concentratwn of the castor-oil soap solution after mixture therewith of the bacterial organisms or of the toxin may var considerably. My experience shows that 1t and 20.0%, and is may be injected without producing death,

to 3.0%. The surfaceand even without; producing material discomfort. Indeed, such a large dose of my modified toxin may be injected without greater ill effects than is ordinarily produced by the small initial dose of the series of doses required to reduce immunity in the use of an unm' 1fied toxin. Yet the treated organisms or toxin retain suflicient stimulating power to serve. as antigens, so

that they incite in the inoculated animal aproduction of anti-bodies; which give the animal immunity against the corresponding disease. y 4

The organisms or toxins treated in accordance with my invention may be usedto inoculate human beings or animals to produce active immunity, against the corresponding disease. This may be done with a very few injections, and ordinarily with a single injection: and may be done without danger of producing anaphylaxis or protein sensitization; such as is caused when a toxin is modi fied by some animal serum,as in a toxinantitoxin. I have thus produced active immunity against scarlet fever, diphtheria, and tetanus by the use of modified toxins,

sis

and a instipneumococcic and stre tococcic infectionsby the use of modified acterial tionation of the blood was to produce against streptococcm and pneumococcic in- Y indicates that following stimulation-of the body plasma) obtained from the blood thus rawn may be used either therapeutically or prophylactically; and is administered by suitable lnjection, thus giving passive im munity to the patient. I have thus ob-- tained effective anti-body serums against scarlet-fever, diphtheria, and tetanus, and

fections. r i

The action of the modified orattenuated or detoxified bacterial organisms or toxin in the animal body is due to an adsorption of the fatty-acid salts on the bacterial organisms or on the toxin molecules, or on both, so that upon injection the antigen is at first partially or entirely restrained from exerting its poisonous action, which would otherwise cause a serious reaction, dose were of suflicient size. My experience the injection the antigen is slowly released from this adsorbed fatty-acid salt, so that the antigen has its out over a relatively longer period of time instead of being suddenlv effective, and the resultant longer-continued of the inoculated animal or human being causes such body to build up resistance by rapidly and continuously. creating the corresponding anti-bodies throughout such stimulation following the injection of the such antigen is morewidely effect per unit of areaever, larger animals,

are also used with success,

longer-continued period of modified bacterial organisms or modified toxin. 7 v

Myexperience lso furnishes evidence that the fatty-acid salt, perhaps by its capacity to dialyze, promotes penetration of the antigen (toxin or bacterial organisms or both) mixed with it into the body tissues, so that distributed through the body and thus exerts its antigenie or stimulatory action over a wider area; with'a maintenance of the stimulating below a critical maximum beyond which ill effects and a less proportionate production of anti-bodiesoccur, and with a greater and more rapid total production of anti-bodies because of the increase in area of the stimulation.

I have found rabbits convenient animals to use for producing the anti-bodies. 'Howespecially in largeor even death if the such as sheep or horses,-

neanne scale. production. 7 The attenuated or detoxified bacterial organisms or the modified toxin,.'after being treated as above described, are suitably injected into the animal, as by subcutaneous, intraperitoneal, or intravenous injection. Theremay be several injections at intervals if desired. with gradually increasmg doses; and I have found that for the commercial production of anti-body serum, five inject-ions at intervals of about twentyfour hours, with the doses ranging from ten to one thousand times the normal lethal dose of unmodified bacterial "organisms ortoxin, produce good results. A sufiicient time is allowed for the injected organisms or toxin to incite a production of anti-bodies in the body of the inoculated animal; and then the serum is obtained from the animal in the usual way. If desired, especially in using larger animals, there maybe repeated withdrawals of blood for serum at suitable intervals; and these withdrawals may be interspersed with repeated injections of the modified antigen. 1 I

It is the serum thus obtained which contains the immunological anti-bodies for the production of passive immunity, and for combating the symptoms of the corresponding disease in patients suffering from such disease.

travenous, although it may be subcutaneous or intramuscular. Within a few hours after the injection. ordinarily, the syndrome of the disease forwhich the treatmentis given begins to disappear. A single injection of the serum is often suificient to produce permanent relief. In some cases, however, a single injection is not suflicient, as the symptoms of the disease, after disappearing for a few hours, sometimes recur later. Vhen such symptoms do recur. whether once or more.

another injection of the serum is indicated;

and, if given, again produces relief. Rarely are more than two or three in ections required. 4

' This present application is directed to the generic phases of my invention, but also includes specific claims on the products used in producing diphtheria immunity. Specific claims to. products used in producing immunity from other diseases are presented in separate applications; my copending application Serial No. 84,168. filed January 27, 1926. containing -claims directed specifically to the modified scarlet fevcrantigen. and my copending application Serial No. 52.964.

also'filcd August 27, 1925, containing claims directed specifically to the modified pneumococcic antigen and to the anti-pneumococcic serum and process of producing it.

In the following claims, in defining the The injection of the serum is desirably inmaterial which is treated. and in defining one i of thecomponents of the mixture, I use the expression bacteria, and/or their products eanne or toxins, to denote either the bacteriaalone, whether livingor dead, or the products or toxins alone of said bacteria, or a mixture of such bacteria (living or dead) and the prod ucts 01' toxins produced thereby.

I claim as my invention: 1. The process of treating bacteria, and/or their products and toxins. to render them non-disease-producing and non-poisonous, which consists in applying thereto surface tension depressants consisting of soluble salts of the higher fattv acids which are adsorbed on the surfaces of the bacteria and the toxin molecules, substantially as herein described. 2. The process of treating bacteria, and/or their products and toxins, to render them non-disease-producing and non-polsonousl, which consists in applying thereto surface tension depressants consisting of salts of the higher unsaturated fatty acids, which are adsorbed on the surfaces of the bacteria and the toxin molecules, substantially as herein described.

3. The process of treating bacteria, and/or their products and toxins. to render them -non-disease-producing and non-poisonous,

which consists in applying thereto surface tension depressants consisting of the salts of the higher unsaturated fatty acids containing one or more hydroxyl groups which are adsorbed on the surfaces of the bacteria and the toxin molecules, substantially as herein described.

4. The process of treating bacteria. and /or their products and toxins, to render them non-disease-producing ,and non-poisonous. which consists in applying thereto eastor oil soap. 7 V v .5. The process of treating bacteria, and/or their products or toxins, to increase the amount thereof which may be injected on a first immunizing dose, comprising treating said bacteria, and/or their products or toxins, with a soluble salt of 'an organic acid which is a surface-tension depressant.

6. The process of treating bacteria, and/or their products or toxins, to increase the amount thereof which may be injected on a first. immunizing dose, comprising treating said bacteria, and/or their products or toxins, with 1 surface-tension depressant comprising a soluble salt of a higher fatty acid.

7. The process of treating bacteria, and/or: their products ortoxins, to increase the amount thereof which may be injected on a first immunizing dose, comprising treating said bacteria. and/or their'products or toxins,

with a surface-tension depressant comprismg a soluble salt of a higher unsaturated fatty acid.

8. The process of treating bacteria, and/or their products or toxins, to increase the amount thereof which may be injected on a first immunizing dose, comprising treating said bacteria, and /or their products or toxins,

with a surface-tension depressant comprismga soluble salt of a higher fatty acid contaming one or more hydroxyl groups.

11. The process of treating a bacterial" toxin to increase the amount thereof which may be in ected on a first immunizing dose,

comprising treating'said toxin-with castor' oil soap.

12. The process of producing an immunizing anti-body serum, comprising treating bacteria, and/or their products or toxins, as set forth inclaim 1, injecting said treated material into an animal, and obtaining blood serum from said animal.

. 13. The process of producing an immunizing anti-body serum, comprising treating bacteria, and/or their products or toxins, as set forth in claim 2, injecting said treated material into an animal, and obtaining blood serum from said animal.

14. The process of producmg an immunizing anti-body serum, comprising treating bacteria, and/or their products or toxins, as

set forth in claim 3, injecting said treated material into an animal, and obtaining blood serum from said animal.

15. The process of producing an immunizing anti-body serum, comprising treating" bacteria, and/or their products or toxins, as

set forth in claim 4, Injecting said treated material into an animal. and obtaining blood serum from said animal.

16. The process of producing an immunizing anti-body serum, comprising treating bacteria, and/or their products or toxins,

with a soluble salt of an organic acid which such treated material'into an animal; and obtaining blood serum from said animal.

17.' The process of producing an immunizing anti-body serum, comprising treating bacteria, and/or their products or toxins,

with a surface-tension depressant comprising a soluble salt of a higher fatty acid; in-

jecting such treated material into an animal; and obtaining blood serum from said animal.

is a surface-tension depressant; injecting 18. The process of producingan immunizing anti-body serum, comprising treating bacterla, and/or .their products ortoxins. w th a surface-tenslon depressant compris- 3 into an anima I jecting such treated material into an animal;

' such treated toxin into an animal; and 0band obtaining blood serum from said animal.

20. The process of producing an immuniz- .ing anti-bodiy serum com rising treating bacteria, an r their pro ucts or toxins, with a surface-tension depressant comprising a soluble salt of a higher unsaturated fatty acid containing one or more h droxyl groups; injecting such treated materia into an animal and obtaining blood serum from said animal.

21. The process of producing an immunizing anti-body serum, com rising treating bacteria, and/or their pro ucts or toxins, with a soluble salt of ricinoleic acid; injecting such treated material into an animal; and obtaining blood serumfrom said animal.

22. The process of producing an immunizing anti-bod serum, comprising treating bacteria, and or their products or toxins, with castor oil soap; in ecting such treated material into an animal; and obtaining blood serum from said animal- 23. The process of treatin diphtheria toxin to increase the amount thereof which may be injected on a first immunizing dose,

comprisin treating said toxin with a solu- 'ble salt 0 an organic acid which is a surface-tension depressant.

24. The process of treating diphtheria toxin to increase the amount thereof which may be injected on a first immunizing dose, comprising treating said toxin with castor oil soap.

25. The process of producing anti-diphtheria serum, I comprising treating diphtheria toxin with a soluble salt of an organic acid which is a surface-tension depressant; injecting such treated toxin into an animal; and obtaining blood serum from said animal. 26. The process of producing anti-diph .theria serum, comprising treating diphtheria toxin with castor oil soap; injecting taining blood serum from said animal. I v

27. Immunizing material consisting of bacteria, and/or their products or toxins,

rendered non-disease-producing and nonpoisonous by having-applied thereto salts of organic acids which are surface-tension depressants and which are adsorbed on to the surface of the bacteria and/or toxin molecules.

28. Immunizing material consisting of bacteria. and/or their products or toxins,

rendered non-disease-producing and nonbacteria;i and/or their products or toxins rendere non-disease-producing and nonoisonous by having applied thereto surace-tension depressants consistin of salts of the higher unsaturated fatty acids, which are adsorbed on the surfaces of the bacteria and/or the toxin molecules, substantially as herein described.

a e o 30. Immumzmg material consisting of bacteria, and/or their products or toxins, rendered non-disease producing and nonpoisonous by having applied thereto surfacetension depressants 'consisting'of salts of the higher unsaturated fatty acids containing one or more hydroxyl groups, which are adsorbed on the surfaces of the bacteria and/or the toxin molecules, substantially as herein described.

31. A composition of matter, comprising a mixture of bacteria. and/or their products or toxins and a soluble salt of an organic acid which is a surface-tension depressant.

32. A composition of matter, comprising a mixture of bacteria, and/or their products or toxins and a surface-tension depressant comprising a soluble salt of a higher fatty acid. 7 33. A comppsition of matter,-comprising a m1xture of acteria, and/or their products .or toxins and a surface-tension de ressant comprising a soluble salt of a big er un saturated fatty acid.

3%. A composition of matter, comprising a m1xture of bacteria, and/or their products or toxins and a surface-tension depressant 38. A composition of matter, comprising a mixture of diphtheria toxinand ,a soluble acterial, and/or their products salt of an organic acid which is a surfacetension depressant.

'39. A composition of matter, comprising a mixture of diphtheria toxin and a surface tension depressant comprising a soluble salt of a higher fatty acid.

40. A composition of matter, comprising a mixture of diphtheria toxin and a surfacetension depressant comprising a soluble salt of a higher unsaturated fatty acid.

41. A composition of matter, comprising a mixture of diphtheria toxin and a surface-' tension depressant comprising a soluble salt of a higher fatty acid containing one or more hydroxyl groups.

42. A composition of matter, comprising a mixture of diphtheria toxin and a surfacevtension depressant comprising a soluble salt of a higher unsaturated fatty acid containing one or more hydroxyl groups.

43. A composition of matter, comprising a mixture of diphtheria toxin and a soluble salt of ricinoleic acid. f 44. A composition of matter, comprising a mixture of diphtheria toxin and castoroil' soap. 1

45. Serums for therapeutic use obtained from animals which have received injections of bacteria, and/or their products and toxins, previously treated with salts of the higher fatty acids, substantially as herein of bacteria, and/or their products and toxins, previously treated with salts of the higher unsaturated fatty acids containing one or more hydroxyl groups, substantially as described. a

49. An anti-diphtheria serum obtained from an animal which has received one or more injections of diphtheria toxin previousl treated with a soluble salt of an organlc acid which is a surface-tension depressant.

50. An anti-diphtheria serum .obtained from an animal which has received one or more injections of diphtheria toxin previously treated with a surface-tension depressant comprising a soluble salt of a higher fatty acid.

51. An anti-diphtheria serum obtained from an animal which has received one or more injections of diphtheria toxin pre viously treated with a surface-tension depressant comprising a soluble salt of a higher unsaturated fatty acid.

. An anti-diphtheria serum obtained fr 111 ananimal which has received one or more injections of diphtheria toxin previously treated with, a surface-tension depressant comprising a soluble salt of a higher fatty acid containing one or more hydroxyl groups. I

53. An anti-diphtheria serum obtained from an animal which has received one or more injections of diphtheria toxin. pre-' viously treated with a surface-tension depressant comprising a soluble salt of a higher unsaturated fatty acid containing one or more hydroxyl groups.

54. An anti-diphtheria serum obtained from an animal which has received one or more injections of diphtheria toxin previously treated with a soluble salt of ricinoleic acid.

55 An antidiphtheria serum obtained 7 from an animal which has received one or more injections of diphtheria toxin previously treated with castor oil soap. 7

In witness whereof, I have hereunto set my hand this 31st day of October, 1925.

WIN FORD P. LARSON. 

